Immunoglobulins are high molecular weight proteins with the capacity to bind specific ligands. Although immunoglobulins are generally thought of as binding agents, they are also capable of being bound, i.e., one immunoglobulin may function as a ligand for another immunoglobulin which functions as a binding agent.
Immunoglobulins from a first species can serve as antigens when injected into a second species. The antiserum raised in the second species contains immunoglobulins directed against antigenic sites or determinants on the immunoglobulins from the first species. The antiserum and immunoglobulins contained therein are said to be heterologous with respect to the first species. Immunoglobulins from the same species are said to be homologous, while immunoglobulins from the same individual are said to be isologous.
The immunoglobulins from any given species can be divided into three broad categories based upon antigenic heterogeneity. Isotypes are those immunoglobulins which share a common effector function, e.g., immunoglobulin G is responsible for humoral immune response, while immunoglobulin E is responsible for the allergic response. The antigenic heterogeneity resides in the type of heavy chain out of which the immunoglobulin is formed. In the examples given, immunoglobulin G comprises a .gamma. chain, while immunoglobulin E comprises an .epsilon. chain.
Although all isotypes share a common function, small differences have been observed within a given isotype of different members of the same species. These differences, which are genetic in origin, account for the second category of immunoglobulins, allotypes.
The final category of immunoglobulin is the idiotype. Because an immunoglobulin of a given isotype and allotype is capable of binding any one of countless ligands with remarkable specificity, it is clear that the ligand binding region of an immunoglobulin directed against one ligand must be different from the ligand binding region of an immunoglobulin directed against another ligand. The difference is reflected in the amino acid sequence of the ligand binding region. Idiotypes are antibodies which are distinguishable on the basis of the chemical nature of at least their ligand binding regions and, therefore, on the basis of the chemical nature of the ligands which they are capable of binding. Idiotypes were first discovered by Oudin who injected anti-Salmonella antibody from a donor rabbit into an allotype-matched, non-immune recipient rabbit. Some recipient rabbits produced antibody directed against the anti-Salmonella antibody. [Oudin, J. and Michel, M., J. Exp. Med., Volume 130, 595, 619 (1969)].
Other researchers found that the idiotypic region comprises the ligand binding site, but that the two are not necessarily coterminus. See, for example, Brient et al., P.N.A.S. Volume 68, 3136 (1971) and Sher et al., J. Immunol., Volume 109, 176 (1972). This conclusion is based on the fact that a reaction of idiotype and anti-idiotype antibodies is, in many cases, only partially inhibitable by the ligand for which the idiotype is specific. As used herein, the term "idiotype" refers to the specific region of an immunoglobulin which imparts its idiotypic character.
With the advent of the radioimmunoassay (RIA) [Yalow and Berson, J. Clin. Invest., Volume 39, 1157 (1960)], the immunoassay became recognized as an exquisitely sensitive tool in the measurement of clinically important substances found at low concentrations in various body fluids.
U.S. Pat. No. 3,654,090 issued to Schuurs on April 4, 1972 teaches the use of an enzyme-substrate system to replace the radioactive label used in the RIA.
Both of these assays make use of a competitive reaction between a limited number of antibody molecules and both a labeled antigen and unlabeled antigen (either known amounts used to construct a standard curve or unknown amounts contained in test samples). The more unlabeled antigen in the reaction mixture, the less labeled antigen will be bound to the limited number of antibody molecules. One must be equipped with a method to detect the ratio of bound to free labeled antigen.
The earliest methods used a physical separation of antibody-bound labeled antigen from free labeled antigen. This type of assay, in which a separation step is employed, is referred to as a heterogeneous assay.
Another method to detect the extent of binding of labeled antigen makes use of a preexisting signal produced by the binding of antibody and labeled antigen which is then modulated, either raised or lowered, by the binding of the unlabeled antigen. Such methods are referred to as homogeneous. U.S. Pat. No. 4,233,402, issued Nov. 11, 1980 to Maggio et al. and U.S. Pat. No. 3,996,345, issued Dec. 7, 1976 to Ullman et al. are illustrative of such methods which may include enzyme-channeling and fluorescence quenching.
The assays referred to above all make use of antigen or hapten (a low molecular weight substance which is not immunogenic but is capable of being bound by specific anti-hapten antibodies) which is identical or immunochemically analogous to the analyte (the unknown being tested for). The antigen or hapten is labeled with reagent means for determining the extent to which the labeled antigen or hapten is bound to the antibody. The use of such substances presents problems in the various assay systems already developed in the art. For example, many antigens, especially bacterial and viral antigens, are difficult to isolate in pure form. Isolation of such antigens can pose significant health hazards. Some antigens or haptens may lack suitable functional groups for the attachment of label. Furthermore, it is often difficult to label an antigen or hapten without altering its structure to the point where it no longer competes for anti-analyte antibody as well as unlabeled antigen; this is especially true for haptens. Labeling an antigen or hapten may also cause instability and susceptibility to rapid degradation. Finally, immobilization of an antigen or hapten on a solid support may decrease its ability to bind to antibody. Consequently, there is a need for a ligand which can act as a functional substitute for antigen or hapten and which
(1) can be safely isolated in large quantities without the need for laborious purification steps;
(2) offers multiple possible sites for the attachment of label;
(3) can be labeled without substantially diminishing its ability to compete for antibody;
(4) can be labeled without substantially diminishing its stability; and
(5) can be bound to a solid support without substantial loss of immunoreactivity.
Although anti-idiotype antibodies have been known in the art for over a decade, their practical utility as ligands in clinical immunoassays has gone unrecognized. It is precisely the use of such anti-idiotype antibodies, primarily as labeled ligands, particularly in homogeneous assays, which forms the basis of the current invention.